Novel aliphatic compounds, process for their preparation and their usage

ABSTRACT

The present invention provides an aliphatic compound represented by the following formula (I) or pharmacologically acceptable salts thereof:  
                 
 
     where n denotes an integer of 1 to 11, and 1 denotes an integer of 1 to 16,  
     the aliphatic compound being an optical isomer of the (2R,3S,2′S) configuration when the 8-position thereof is a double bond, or an optical isomer of the (2S,3R,2′RS) configuration when the 8-position is a single bond; methods for producing the compound or pharmacologically acceptable salts thereof; and uses of the compound in the treatment of cardiovascular diseases (e.g. arteriosclerosis, cardiac diseases), cancer, rheumatism, diabetic retinopathy, and respiratory diseases.

TECHNICAL FIELD

[0001] This invention relates to novel aliphatic compounds, methods forproducing them, and pharmaceuticals comprising the aliphatic compoundsas an active ingredient.

BACKGROUND ART

[0002] Alpha granules released from activated platelets during theprocess of hemostasis contain serotonin, ADP and the aliphaticderivative, 2-amino-3-hydroxy-4-octadecene-1-phosphate (AHOP). Serotoninshows vasoconstriction, and ADP exhibits platelet aggregation, bothcompounds promoting hemostasis, whereas the role of AHOP has beenunknown. In recent years, endothelial differentiation gene (Edg), anorphan receptor for which AHOP is an endogenous ligand, has beendiscovered. The possibility is being shown that the binding of AHOP andEdg acts in directions toward promotion of arteriosclerosis, such ashemodynamic aggravation or vascular smooth muscle growth, or indirections toward the progression of respiratory diseases.

[0003] The gene of Edg was cloned as an orphan receptor in 1990 [Edg-1(JBC, '90, 265, p. 9308)]. Then, Edg-3 (BBRC, '96, 227, p. 608) andEdg-5 (AGR16/H218) (JCB, '96, 135, p. 1071) were obtained as homologuesof Edg-1, but their physiological roles remained unclear. In 1998,however, the possibility of AHOP being an endogenous ligand for Edg-1was suggested (Science, '98, 279, p. 1552), and then Edg-3 and Edg-5were also shown to be AHOP-specific receptors (BBRC, '99, 260, p. 263;JBC, '99, 274, 27, p. 18997).

[0004] Edg-1 on the vascular endothelial cell, when stimulated by AHOP,upregulates an adhesion protein, such as cadherin, through activation oflow molecular weight GTP-binding protein Rho (Science, '98, 279, p.1552). T lymphocyte-derived strain cells, upon stimulation by AHOP,accelerates vascular layer penetration in an in vitro pseudo-bloodvessel model (EMBO J., '98, vol. 17, No. 14, p.4066). Okajima et al.conducted pseudo-blood vessel migration tests using CHO cells forced toexpress Edg-1 or Edg-3, and found migration to be promoted AHOPconcentration-dependently in either case ('99 Congress of the JapaneseBiochemical Society, A collection of the Abstracts, p. 883). On theother hand, Igarashi et al. showed that the cancer cell strain F10underwent suppression of migration by about a maximum of 80%concentration-dependently at an AHOP concentration of 10⁻⁸ to 10⁻⁶ M ina pseudo-blood vessel model, but Edg-1 or Edg-3 was scarcely expressed,and Edg-5 was expressed, in the F10 cells ('99 Congress of the JapaneseBiochemical Society). In connection with these findings, the possibilitywas pointed out that AHOP showed the suppression of migration because ofa difference in subspecies ('99 Congress of the Japanese BiochemicalSociety, A collection of the Abstracts, p. 675, p. 883).

[0005] AHOP-responsive activation of MAP kinase was observed in vascularsmooth muscle cells (Eur. J. Biochem., '98, 257, p. 403) or respiratorytract smooth muscle cells (Biochem. J., '99, 338, p. 643), indicatingthe possibility for AHOP to act in a direction toward the growth ofvascular smooth muscle cells.

[0006] Sugiyama et al. administered AHOP to rats by the caudal veinroute, and observed hemodynamics. They noted significant drops in twoparameters, systolic blood pressure and time differential of leftventricular pressure, showing the possibility that AHOP acts in adirection toward decline of cardiac function in vivo (A collection ofthe Abstracts at the '00 Congress of the Japanese PharmacologicalSociety, p. 127).

[0007] The possibility is also pointed out that AHOP activatesmuscarinic receptor inward K⁺ rectifier to cause arrhythmia ('99 PfugersArch-Eur J Phisiol 438, pp. 642-648). Thus, an Edg receptor antagonistcan be considered to have a possibility for taking effect againstarrhythmia.

[0008] The effect of AHOP on vascular endothelial cells was studied inan angiogenic animal model. This study demonstrated that angiogenesis bya growth factor, such as VEGF or FGF-2, was synergistically promoted byAHOP bound to Edg-1 or Edg-3, thus showing the possibility that Edg actson the progression of rheumatism, solid carcinoma, or diabeticretinopathy (Cell, '99, p. 301).

[0009] The possibility has been presented that excessive inflammation orrespiratory tract remodeling, caused by the binding of AHOP and Edgreceptor, results in the progression of pneumonia, chronic obstructiveairway disease, COPD) or respiratory hypertension (PulmonaryPharmacology & Therapeutics, 2000, 13, p. 99).

[0010] Suramin, an agent for eradicating Protozoa Trypanosoma, isreported to show Edg-3-specific antagonism and inhibit a signal forbinding of AHOP and Edg (J. B. C., '99, 274, 27, p. 18997). Suramin isshown to be therapeutically effective in arteriosclerosis pathogenesismodels (Circulation, '99, Cardiovascular Res., '94, 28, p. 1166), andEdg antagonism may be involved in the mechanism of this therapeuticeffect.

[0011] Considered overall, these findings show the possibilities thatAHOP bound to Edg acts in promoting arteriosclerosis, as evidenced byinflammatory cell activation, vascular smooth muscle cell growth orhemodynamic aggravation, and in promoting angiogenesis in favor ofprogression of rheumatism, solid carcinoma, or diabetic retinopathy.That is, substances antagonizing Edg are likely to show the propertiesof anti-cardiovascular diseases (for example, anti-arteriosclerosis,anti-cardiac diseases (e.g. anti-arrhythmia, anti-myocardialinfarction)), anti-rheumatism, anti-cancer, anti-diabetic retinopathy,and anti-respiratory diseases.

[0012] The inventors of this invention performed in-depth studies in thelight of the above circumstances, and newly discovered compoundsrepresented by formulas (I) to (V) shown below. They found that thesecompounds (hereinafter referred to as “compounds of the presentinvention”) are antagonistic to Edg receptor. The present invention isbased on this finding, and its object is to provide novel aliphaticcompounds, methods for producing them, and pharmaceuticals comprisingthese compounds.

DISCLOSURE OF THE INVENTION

[0013] The present invention relates to an aliphatic compoundrepresented by the following formula (I)

[0014] where n denotes an integer of 1 to 11, and 1 denotes an integerof 1 to 16,

[0015] which is an optical isomer of the (2R,3S,2′S) configuration whenthe 8-position is a double bond, or an optical isomer of the(2S,3R,2′RS) configuration when the 8-position is a single bond.

[0016] In the above formula, the wavy line refers to the inclusion ofany of the optical isomerisms (R), (S) and racemic modification. Herein,the upper chain is called the first chain, and the lower chain is calledthe second chain.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]FIG. 1 is a graph showing that the compounds of the presentinvention are Edg-antagonistic dose-dependently (suramin: control).

[0018] In the drawing, an unfilled triangle signifies data obtained whenno test substance is incorporated.

[0019]FIG. 2 is a graph in which the compounds of the present inventionshow an AHOP-competitive action dose-dependently.

[0020] In the drawing, an unfilled circle signifies data obtained whenno test substance is incorporated.

[0021]FIG. 3 is a graph in which the compounds of the present inventionshow the action of suppressing vascular smooth muscle cell growthdose-dependently (suramin: control).

[0022] In the drawing, an unfilled rectangle signifies data obtainedwhen no test substance is incorporated. *: Shows significant suppressionat significance level p≦0.05 against the negative control. **: Showssignificant suppression at significance level p≦0.01 against thenegative control.

[0023]FIG. 4 shows the action of the compounds of the present inventionon endothelial cell-neutrophil interaction. **: Shows significantsuppression at significance level p≦0.01 against the control.

BEST MODE FOR CARRYING OUT THE INVENTION

[0024] Preferred embodiments are cited below.

[0025] The present invention provides the compound of the aforementionedformula (I) which is a compound of the following formula (II):

[0026] Formula (II)

[0027] where n and l have the same meanings as those of the symbols inthe compound of the formula (I).

[0028] The present invention also provides the compound of theaforementioned formula (I) which is a compound of the following formula(III):

[0029] Formula (III)

[0030] where n and l have the same meanings as those of the symbols inthe compound of the formula (I).

[0031] In the compounds of the formulas (I), (II) and (III) of thepresent invention, preferably n is 1 to 10 and l is 1 to 15, and morepreferably n is 1 to 8 and l is 1 to 13.

[0032] The present invention also provides the compound of theaforementioned formula (I) which is a compound of the following formula(IV):(4E,8E,2R,3S,2′S)-N-2′-hydroxyhexadecanoyl-9-methyl-4,8-octadecadiene-1,3-diol

[0033] Formula (IV)

[0034] The present invention also provides the compound of theaforementioned formula (I) which is a compound of the following formula(V):(4E,2S,3R,2′RS)-N-2′-hydroxyhexadecanoyl-9-methyl-4-octadecene-1,3-diol

[0035] Formula (V)

[0036] The compounds of the present invention can form pharmacologicallyacceptable salts thereof. The salts are not limited, and include, forexample, hydrohalogenic acid salts, such as hydrofluorides,hydrochlorides, hydrobromides and hydroiodides, inorganic acid salts,such as nitrates, perchlorates, sulfates, phosphates, and carbonates,lower alkylsulfonic acid salts, such as methanesulfonates,trifluoromethanesulfonates and ethanesulfonates, arylsulfonic acidsalts, such as benzenesulfonates and p-toluenesulfonates, carboxylicacid salts, such as acetates, fumarates, succinates, citrates,tartrates, oxalates and maleates, amino acid salts, such as glycinesalts, alanine salts, glutamates and aspartates, and alkali metal salts,such as sodium salts and potassium salts.

[0037] The compounds of the present invention all show endothelialdifferentiation gene (Edg) receptor antagonism, antagonize the bindingof Edg receptor agonizing substances, such as AHOP andsphingosylphosphorylcholine, to Edg receptors, and can inhibit theintracellular signal transduction system relying on these substances.

[0038] Hence, the present invention provides pharmaceuticalsantagonizing endothelial differentiation gene (Edg) receptor, thepharmaceuticals comprising the compounds of the formulas (I) to (V) asan active ingredient.

[0039] Moreover, the present invention provides the pharmaceuticals fortreating diseases resulting from the activation of inflammatory cells,the growth of vascular smooth muscle cells, the aggravation ofhemodynamics, and angiogenesis, for example, cardiovascular diseases(e.g. arteriosclerosis, cardiac diseases (e.g. myocardial infarction,arrhythmia)), rheumatism (e.g. rheumatoid arthritis), cancer, diabeticretinopathy, and respiratory diseases (e.g. pneumonia, chronicobstructive airway disease, respiratory system hypertension).

[0040] The “treatment” includes prevention as well.

[0041] The “cardiovascular diseases” refer to diseases in which thecirculatory state of blood or lymph is disturbed, resulting in disorderof a tissue or cells. Their examples are arteriosclerotic diseases (e.g.atherosclerosis), and cardiac diseases (e.g. myocardial infarction,arrhythmia).

[0042] The “respiratory diseases” refer to diseases, in which therespiratory organ, such as trachea, bronchus or lung, is disordered, andsymptoms related to them. Their examples are asthma (immediate, delayedor allergic asthma), bronchial asthma, allergic rhinitis, eosinophilicinfiltration, bronchitis (chronic bronchitis), respiratory tractinflammation, pulmonary emphysema, pneumonia, chronic obstructivepulmonary disease (COPD), acute respiratory distress syndrome,respiratory hypertension, dyspnea, pain, coughing, sputum, vomiting, andshortness of breath.

[0043] For use as pharmaceuticals, the compounds of the presentinvention may be in any forms, such as solid compositions, liquidcompositions and other compositions, and optimal forms are selectedaccording to needs. Pharmaceutical compositions can be prepared indosage forms, such as tablets, pills, capsules, granules, powders,liquids and solutions, emulsions, suspensions, and injections, by addingexcipients, bulking agents, binders, disintegrants, pH adjustors andsolubilizers, which are in customary use, to the compounds of thepresent invention, and treating the mixtures by customary pharmaceuticalmanufacturing techniques. Examples of the excipients and the bulkingagents are lactose, magnesium stearate, starch, talc, gelatin, agar,pectin, acacia, olive oil, sesame oil, cacao butter, ethylene glycol,and other materials which are commonly used.

[0044] To prevent the oxidation of the resulting preparations,antioxidants (e.g. tocopherol) may be added, the preparations may beincluded with inclusion agents, such as cyclodextrin, or thepreparations may be encapsulated with a film of gelatin or the like.

[0045] Furthermore, the aforementioned compounds can be produced as O/Wpreparations, as described in Japanese Unexamined Patent Publication No.6-298642, with the use of phospholipids or nonionic surfactants asemulsifying agents. The emulsifying agents can be used alone or incombination of two or more, and the amount of the emulsifying agent maybe 0.001 to 10% (W/V), as desired, or preferably 0.01 to 5% (W/V).

[0046] Examples of the phospholipids are soybean-derived phospholipid,egg yolk-derived phospholipid, lysolecithin, phosphatidylcholine(lecithin), and phosphatidylserine, which can be used alone or incombination. Examples of the nonionic surfactants are, but not limitedto, polyoxyethylene-polyoxypropylene block copolymer with a molecularweight of 500 to 15,000 (e.g. Pluronic F-68), polyalkylene glycol with amolecular weight of 1,000 to 10,000, polyoxyalkylene copolymer with amolecular weight of 1,000 to 20,000, hydrogenated castor oilpolyoxyalkylene derivatives, castor oil polyoxyalkylene derivatives,glycerin fatty acid esters, polyglycerin fatty acid esters, sorbitanfatty acid esters, polyoxyethylene castor oil, hydrogenated castor oil,polyoxyethylene alkyl ethers, and sucrose fatty acid esters, which arepreferably used alone or in combination.

[0047] The compounds of the present invention can be administered orallyor parenterally in a dose of about 0.0001 to about 100 mg/kg bodyweight/day, which was given once daily or in several divided doses perday. This dosage can be increased or decreased appropriately dependingon the type of the disease, or the age, body weight or symptoms of thepatient.

[0048] The compounds of the present invention can be produced by thefollowing methods of production:

SYNTHETIC EXAMPLE 1

[0049] The method of producing the compound of the formula (I) will bedescribed, including a Preparation Example for starting materials forreactions.

[0050] (1) Preparation Example for Reaction Materials

[0051] (A) Synthesis of Oxazoline Aldehyde Derivative

[0052] An oxazoline aldehyde derivative of the following formula can besynthesized by a conventional method:

[0053] where R′ represents an alkyl group or an aryl group, and R′ ispreferably an aryl group (for example, a phenyl group).

[0054] For example, when (L)-serine is used as a starting material, thecompound can be produced in the following manner:

[0055] (L)-serine is converted to its ester (e.g. Me ester) (Ber. Dtsch.Chem. Ges. 39, 2949(1906)). The serine used here is R-serine if theoptical isomerism of the first chain of the desired compound correspondsto 2R-isomer, or S-serine if the optical isomerism of the first chain ofthe desired compound corresponds to 2S-isomer.

[0056] Then, the resulting serine ester is reacted using an imino ether(e.g. benzimino ethyl ether) under Elliott's conditions (J. Chem. Soc.589 (1949)) to obtain an oxazoline ester derivative. Since the oxazolineester formed here retains the same optical isomerism as the startingserine and shows no racemization, it is advantageous in obtaining thedesired optical isomer.

[0057] Then, an oxazoline aldehyde derivative is obtained from theresulting oxazoline ester derivative.

[0058] This reduction reaction is performed in an inert solvent (e.g.hexane) in the copresence of a metal hydride (e.g. DIBAL-H(diisobutylaluminum hydride)). After the reaction is terminated, thereaction mixture is extracted with a solvent (e.g. an aqueous solutionof sodium potassium tartrate, and EtOAc), whereby the desired oxazolinealdehyde derivative can be obtained.

[0059] Since the resulting oxazoline aldehyde derivative is unstable, itis preferably subjected immediately to a reaction at a subsequent stage.

[0060] (B) Synthesis of (E)-Form Alkenylalane

[0061] An alkenylalane of the following formula can be synthesized inthe conventional manner:

H₃C—(CH₂)_(n)—C(CH₃)

C(H)—(CH₂)₂—CH═CH—Al(R)₂

[0062] where n is as defined for the compound of the formula (I), and Rrepresents an alkyl group, preferably i-Bu.

[0063] For example, the desired alkenylalane in which R is i-Bu can beobtained by the addition reaction of DIBAL-H with alkyne (J. Am. Chem.Soc. 95, 4098 (1973)).

[0064] This reaction can be performed in an inert solvent (e.g. hexane)at a temperature of 20 to 50° C.

[0065] Since the alkenylalane is unstable, it is preferably used, in theform of the resulting product, for a subsequent step. The alkenylalanecan be easily confirmed by the substances used in the formation step andthe product of the subsequent step.

[0066] The alkyne, the starting material for synthesis of thealkenylalane, can be synthesized by various methods, which include, forexample, the following method:

[0067] An alcohol compound is converted into a bromide compound by anordinary method via a tosylate. The bromide compound is converted into anitrile compound, and further reduced in the copresence of a metalhydride (e.g. DIBAL-H) to obtain an aldehyde compound. Then, thealdehyde is converted into a dibromoalkene by the method of Corey et al.(Tetrahedron Lett. 3769 (1972)) using Ph₃P and CBr₄, whereafter analkyne is synthesized in the presence of a strong base (e.g. n-BuLi).

[0068] (2) Synthesis of the Compound of the Formula (I)

[0069] A synthesis scheme for the compound of the formula (I) isindicated below.

[0070] First step: The oxazoline aldehyde derivative obtained in (1)(A)is alkenylated with the alkenylalane obtained in (1)(B) to obtain adiastereomer mixture. The oxazoline aldehyde derivative used here is onehaving the same optical isomerism as that at the 2-position of the firstchain of the desired compound (if the desired compound is 2R-isomer, anR-compound is used, and if the desired compound is 2S-isomer, anS-compound is used).

[0071] This reaction can be performed in an inert solvent (e.g. ether)at a temperature of −5 to 10° C.

[0072] The diastereomer mixture formed by this reaction contains twocompounds in which the optical isomerism of the OH group formed by thereaction between the oxazoline aldehyde derivative and the alkenylalaneis in the R- and S-configurations. Thus, it is preferred to separate thediastereomer corresponding to the desired compound (if the opticalisomerism at the 3-position of the first chain of the desired compoundcorresponds to 3R-isomer, the R-compound is separated, and if thedesired compound is 3S-isomer, the S-compound is separated).

[0073] Separation of the diastereomer can be carried out using ordinarychromatography.

[0074] Second step: The above product is ring-opened at its oxazolinering to obtain a compound having an NH₂ group and an —OC(═O)R′ group.Ring opening can be performed in the presence of an acid (e.g. dilutedHCl).

[0075] Third step: After ring opening, the product is selectivelyN-acylated with an acylating agent having the same optical isomerism asthat at the 2′-position of the second chain of the desired compound.Then, a —C(═O)R′ group is eliminated, whereby the compound of theformula (I) can be synthesized.

[0076] As the acylating agent, there can be used an ester (e.g.p-nitrophenyl ester) of H₃C—(CH₂)₁—CH(OH)C(═O)—OH (where l is as definedfor the compound of the formula (I)). The hydroxyl group of theacylating agent is preferably protected (e.g., with acetyl (Ac)).

[0077] The selective N-acylation can be performed in a basic solvent(e.g. pyridine) at a temperature of 30 to 45° C.

[0078] The elimination of the —C(═O)R′ group can be performed using abase (e.g. NaOH), and if the hydroxyl group is protected with Ac asmentioned above, the Ac group can also be eliminated simultaneously withthis elimination.

SYNTHETIC EXAMPLE 2

[0079] The method of producing the compound of the formula (II) will bedescribed, including a Preparation Example for reaction materials.

[0080] (1) Preparation Example for Reaction Materials

[0081] (A) Synthesis of HC≡C—(CH₂)₂—CH═C(CH₃)—(CH₂)_(n)CH₃ in (E)-Form

[0082] (E)-6-methyl-5-pentadecen-1-yne with n=8 will be taken as anexample for the purpose of explanation. The captioned compound havingother definition for n (n is as defined for the compound of the formula(II)) can be synthesized in the same manner as in reactions (to bedescribed below) by using H₃CC(═O)(CH₂)_(n)CH₃ instead of 2-undecanone.

[0083] The method mentioned in Synthetic Example 1 can be used, but herethe following method is used:

[0084] By subjecting 2-undecanone to Horner-Wittig reaction, a geometricisomer of methyl 3-methyl-2-dodecenoate is obtained.

[0085] This ester is alcoholized in the presence of a metal hydride(e.g. LiAlH₄), and thereby obtained as an E/Z mixture containing analcohol, i.e. (E)-3-methyl-2-dodecen-1-ol. This mixture is subjected tosilica gel column chromatography to separate the (E)-isomer.

[0086] Then, the hydroxyl group of the (E)-isomer is substituted bybromine to obtain (E)-1-bromo-3-methyl-2-dodecene. This reaction can beperformed under reaction conditions for substituting a hydroxyl group bybromine. For example, the reaction can be performed by causing bromineto act on the (E)-isomer in an inert solvent (e.g. acetonitrile) in thepresence of phosphine (e.g. triphenylphosphine).

[0087] Then, (E)-1-bromo-3-methyl-2-dodecene is reacted with a Grignardreagent prepared from a propargyl halide (e.g. propargyl bromide) toobtain (E)-6-methyl-5-pentadecen-1-yne. This reaction can be performedin an inert solvent (e.g. diethyl ether) at a temperature of 0 to 5° C.in the presence of a catalyst (e.g. CuCl).

[0088] (B) Synthesis of N-Protected (R)-Formyloxazolidine Derivative

[0089] An N-protected (R)-formyloxazolidine derivative of the followingformula can be synthesized by the conventional method. For example, itcan be synthesized from (R)-serine by the method of Mori et al.(Tetrahedron 1985, 41, 2379-2386).

[0090] where A represents a protective group for N, and B and C eachrepresent an alkyl group (e.g. a methyl group).

[0091] Examples of the protective group A for N are groups, such asbenzyloxycarbonyl (Z), t-butoxycarbonyl (Boc), t-aminooxycarbonyl (Aoc),isobornyloxycarbonyl, p-methoxybenzyloxycarbonyl,2-chlorobenzyloxycarbonyl, adamantyloxycarbonyl, trifluoroacetyl,phthaloyl, formyl, o-nitrophenylsulfenyl, and diphenylphosphinothioyl.Preferably, Boc is used.

[0092] (2) Synthesis of the Compound of the Formula (II)

[0093] A synthesis scheme for the compound of the formula (II) isindicated below.

[0094] First step: The HC≡C—(CH₂)₂—CH═C(CH₃)—(CH₂)_(n)CH₃ in (E)-formobtained in (1)(A) is reacted with the N-protected (R)-formyloxazolidinederivative obtained in (1)(B).

[0095] The reaction in the first step can be performed in an inertsolvent (for example, THF) at a temperature of −10 to −30° C. in thepresence of a base (e.g. n-butyl lithium).

[0096] Second step: The triple bond of the product in the first step isreduced to an (E)-type double bond. Simultaneously, the oxazolidine isdeprotected while it is ring-opened, to obtain a compound having an NH₂group and an OH group.

[0097] The reaction in the second step can be performed in an inertsolvent (for example, THF) at a temperature of −70 to −78° C. using analkali metal and an amine (e.g. lithium in the presence of ethylamine).

[0098] Third Step:

[0099] (1) The product in the second step is treated with a protectingagent for a hydroxyl group.

[0100] For example, 2,2-dimethoxypropane can be used as the protectingagent for a hydroxy group. This reaction can be performed in an inertsolvent (e.g. trichloromethane) in the presence of an acid catalyst(e.g. pyridinium p-toluenesulfonate).

[0101] (2) Then, the protected compound is reacted with a carboxylicacid compound of the following formula:

[0102] where R″ represents a protective group for OH, and l has the samemeaning as in the compound of the formula (II).

[0103] This reaction can be performed in an inert solvent (e.g. drydichloromethane) in the presence of a dehydration condensation agent(e.g. dicyclohexylcarbodiimide and 1-hydroxybenzotriazole).

[0104] The carboxylic acid compound used here can be synthesized by themethod of Mori et al. (Liebigs Ann. Chem. 1994, 41-48), and anOH-protecting group, e.g., tert-butyldiphenylsilyl (TBDPS), can be namedas R″.

[0105] Fourth step: The protective group for the hydroxyl group issubjected to deprotection, and the R″ group is eliminated.

[0106] Deprotection of the protective group for the hydroxyl group canbe performed in an inert solvent (e.g. CH₂Cl₂ and MeOH) in the presenceof an acid catalyst (e.g. pyridinium p-toluenesulfonate). Elimination ofthe TBDPS can be performed in an inert solvent (e.g. THF) using afluorine anion (e.g. tetra-n-butylammonium fluoride).

SYNTHETIC EXAMPLE 3

[0107] The method of producing the compound of the formula (III) will bedescribed, including a Preparation Example for reaction materials.

[0108] (1) Preparation Example for Reaction Materials

[0109] (A) Synthesis of HO—CH₂—CH═C(CH₃)—(CH₂)_(n)CH₃

[0110] This compound can be obtained from H₃CC(═O)(CH₂)_(n)CH₃ (where nhas the same meaning as in the compound of the formula (III)) in thesame manner as in Synthetic Example 2.

[0111] (B) Synthesis of N-Protected (S)-Formyloxazolidine Derivative

[0112] An N-protected (S)-formyloxazolidine derivative of the followingformula can be synthesized by the conventional method. For example, itcan be synthesized from (S)-serine in the same manner as mentioned inSynthetic Example 2.

[0113] where A represents a protective group for N, and B and C eachrepresent an alkyl group (e.g. a methyl group).

[0114] Examples of the protective group A for N are groups, such asbenzyloxycarbonyl (Z), t-butoxycarbonyl (Boc), t-aminooxycarbonyl (Aoc),isobornyloxycarbonyl, p-methoxybenzyloxycarbonyl,2-chlorobenzyloxycarbonyl, adamantyloxycarbonyl, trifluoroacetyl,phthaloyl, formyl, o-nitrophenylsulfenyl, and diphenylphosphinothioyl.Preferably, Boc is used.

[0115] (2) Synthesis of the Compound of the Formula (III)

[0116] A synthesis scheme for the compound of the formula. (III) isindicated below.

[0117] First step: The unsaturated moiety of theHO—CH₂—CH═C(CH₃)—(CH₂)_(n)CH₃ obtained in (1)(A) is saturated bycatalytic reduction.

[0118] This reaction can be performed using various catalysts commonlyused in catalytic reduction. For example, a palladium catalyst (e.g.palladium-carbon, Pd—C) can be used.

[0119] Second step: The hydroxyl group of the product in the first stepis substituted by bromine.

[0120] Bromination can be performed by a method capable of brominatingan alcohol. For example, bromination can be performed by converting theproduct into a tosylate, and then brominating it.

[0121] In this case, the product can be reacted with p-toluenesulfonylchloride in an inert solvent (e.g. pyridine) to obtain a tosylate, andthen the tosylate can be reacted in the presence of a bromide (e.g.sodium bromide) in an inert solvent (e.g. a dimethylformamide (DMF)solution).

[0122] Third step: The bromine of the product in the second step issubstituted by CH₃—C≡C—.

[0123] A reaction material for use in the substitution by CH₃—C≡C— may,for example, be CH₃—C≡C—Li. In the case of CH₃—C≡C—Li, the reaction inthe third step can be performed, for example, in the following manner:

[0124] To a solution of propyne in an inert solvent (e.g. THF solution),a ligand (e.g. tetramethylethylenediamine (TMEDA)) is added (preferablyunder Ar). To the mixture, an alkyl lithium (e.g. n-BuLi) is added toobtain CH₃—C≡C—Li. The reaction temperature is preferably −78 to 0° C.

[0125] Then, a solution (e.g. a mixture of hexamethylphosphoramide(HMPA) and THF) of the product in the second step is added to the abovereaction mixture, whereby the substitution can be performed. On thisoccasion, the reaction temperature is preferably −78 to 20° C.

[0126] Fourth step: The location of the triple bond of the product inthe third step is shifted to the terminal to obtain a compoundterminated with the triple bond.

[0127] The reaction in the fourth step can be performed, for example, inthe following manner:

[0128] An alkali metal (e.g. lithium) is added to an amine base (e.g.1,3-diaminopropane) (preferably under Ar). This reaction is preferablyperformed at −78 to −70° C.

[0129] Then, a strongly basic alcoholate (e.g. potassium t-butoxide) isadded, and the product in the third step is added, whereby the reactionis performed. This reaction is preferably carried out at 15 to 25° C.

[0130] Fifth step: The N-protected (S)-formyloxazolidine derivativeobtained in (1)(B) is reacted with the product in the fourth step.

[0131] The reaction in the fifth step can be performed in an inertsolvent (e.g. THF) at a temperature of −15 to −28° C. in the presence ofa base (e.g. n-butyl lithium).

[0132] A sixth step and subsequent steps can be performed in the samemanner as in the second and subsequent steps of Synthetic Example 2.

[0133] Sixth step: The triple bond of the product in the fourth step isreduced to an (E)-type double bond. Simultaneously, the oxazolidine isdeprotected while it is ring-opened, to obtain a compound having an NH₂group and an OH group. Seventh step: This compound having the (E)-typedouble bond is treated with a protecting agent for a hydroxyl group, andthe protected compound is reacted with a carboxylic acid compound of thefollowing formula:

[0134] where R″ represents a protective group for OH, and l has the samemeaning as in the compound of the formula (III).

[0135] The carboxylic acid compound used here can be synthesized in thesame manner as in Synthetic Example 2. Eighth step: The protective groupfor the hydroxyl group is subjected to deprotection, and the R″ group iseliminated.

EXAMPLES

[0136] The present invention will be described in further detail byworking examples, which in no way limit the technical scope of theinvention.

Example 1 Synthesis of(4E,8E,2R,3S,2′S)-N-2′-hydroxyhexadecanoyl-9-methyl-4,8-octadecadiene-1,3-diol

[0137] A reaction scheme of Example 1 is shown below.

[0138] (1) Preparation of Reaction Materials

[0139] (A) Synthesis of Alkenylalane (8)

[0140] p-TsCl (45 g, 236 mmols) was added to a stirred and cooledpyridine (120 ml) solution of (E)-4-methyl-3-tridecen-1-ol (1) (33 g,155 mmols). The mixture was stirred for 8 hours. Then, the mixture waspoured into ice water (500 ml), and extracted with ether (500 ml). Theether solution was washed with 2N—HCl, a saturated aqueous solution ofNaHCO₃, and a saturated aqueous solution of sodium chloride, dried(MgSO₄), and concentrated in a vacuum. The residue in crude oily form,(E)-4-methyl-3-tridecene-1-tosylate (2) (58 g), was dissolved in DMF(250 ml).

[0141] To the solution, LiBr (40 g, 460 mmols) was added, and themixture was stirred for 18 hours at room temperature. Then, the reactionmixture was poured into ice water (1 L), and extracted with ether (300ml×3). The ether solution was washed with water, dried (MgSO₄), andconcentrated in a vacuum. The residue was distilled to obtain 38.3 g(93.4%) of (E)-1-bromo-4-methyl-3-tridecene (3).

[0142] A mixture of the (E)-1-bromo-4-methyl-3-tridecene (3) (38.0 g,138 mmols) and KCN (11.5 g, 176 mmols) in DMF (100 ml) and water (30 ml)was stirred for 24 hours at 70° C. Then, the mixture was poured into icewater (1 L) and extracted with ether (500 ml). The ether solution waswashed with water, dried (MgSO₄), and concentrated in a vacuum. Theresidue was subjected to silica gel chromatography, and eluted withn-hexane-ether (100:1) to obtain 30.0 g (98%) of(E)-5-methyl-4-tetradecenenitrile (4) as oily matter.

[0143] To a cooled and stirred ether (700 ml) solution of the(E)-5-methyl-4-tetradecenenitrile (4) (30.0 g, 136 mmols), an n-hexanesolution of DIBAL-H (1.7 M, 123 ml, 209 mmols) was added dropwise at−60° C. under an argon gas. The mixture was stirred for 1 hour at −60°C. and for 3 hours at room temperature. The excess reagent was quenchedwith the addition of HCO₂Et (5 ml). After 30 minutes of stirring, themixture was poured into a saturated aqueous solution of NH₄Cl (1.5 L).The resulting mixture was stirred for 20 minutes, acidified with a 20%aqueous solution (1 L) of H₂SO₄, and extracted with ether. The ethersolution was washed with water, dried (MgSO₄), and concentrated in avacuum. The oily residue was subjected to chromatography on Florisil(450 g), and eluted with n-hexane-ether (50:1) to obtain 29.0 g (95.4%)of (E)-5-methyl-4-tetradecenal (5).

[0144] A dichloromethane solution (100 ml) of CBr₄ (85 g, 256 mmols) wasadded dropwise to a stirred and ice-cooled dichloromethane solution (300ml) of Ph₃P (138 g, 526 mmols). To the mixture, a dichloromethanesolution (100 ml) of the (E)-5-methyl-4-tetradecenal (5) (29.0 g, 129mmols) was added while being cooled at 0° C. and stirred, followed bystirring the mixture for 15 minutes at 0° C. The reaction of thismixture was terminated by ice-cooled water (100 ml), and after 20minutes of stirring, the organic layer was separated. The organicsolution was dried (magnesium sulfate), and then concentrated in avacuum. The residue was triturated with pentane (1 L), and insolublePh₃PO was removed by filtration. The filtrate was concentrated in avacuum, and then the oily residue was subjected to silica gel columnchromatography, and eluted with n-hexane to obtain 38.1 g (77.6%) ofoily (E)-1,1-dibromo-6-methyl-1,5-pentadecadiene (6).

[0145] To a stirred and cooled THF (400 ml) solution of the(E)-1,1-dibromo-6-methyl-1,5-pentadecadiene (6) (37.0 g, 97.6 mmols), ann-hexane solution of n-BuLi (1.5 M, 150 ml, 225 mmols) was addeddropwise at −70° C. under an Ar gas. The mixture was stirred for 1 hourat −70° C. and for 1.5 hours at room temperature. Then, the reactionmixture was poured into 1.5 liters of ice water, and extracted withn-hexane. The hexane solution was washed with water, dried with sodiumsulfate, and concentrated in a vacuum. The residue was subjected tosilica gel column chromatography, and eluted with n-hexane to obtain18.9 g (88.0%) of (E)-6-methyl-5-pentadecen-1-yne (7) as oily matter.

[0146] To a stirred n-hexane (5 ml) solution of the(E)-6-methyl-5-pentadecen-1-yne (7) (1.4 g, 6.4 mmols), a solution ofDIBAL-H (1.7 M, 3.8 ml, 6.4 mmols) in n-hexane was added dropwise in thepresence of an Ar gas. The mixture was stirred for 2 hours at 50° C.,and the resulting solution of alkenylalane (8) was cooled with an icebath.

[0147] (B) Synthesis of (R)-4-formyl-2-phenyl-1.3-oxazolin-2-en

[0148] An HCl gas was vigorously bubbled in a dry MeOH solution of(R)-serine (9) (25 g, 238 mmols) until the solution became very hot(spontaneous reflux). The solution was allowed to stand for 16 hours atroom temperature, and then MeOH was removed under vacuum. The residuewas triturated with ether (50 ml). The resulting (R)-serine Me ester(10) in solid form was recovered on a filter paper, washed with ether(50 ml), and dried in a vacuum. Recrystallization from MeOH-ether (1:3)gave 35.9 g (97.0%) of (R)-serine Me ester (10).

[0149] A solution of PhC(═NH)OEt (60 g, 0.4 mol) in dichloromethane (100ml) was added to an aqueous solution (20 ml) of the (R)-serine Me ester(10) in HCl (33 g, 0.21 mol). The mixture was vigorously stirred for 24hours at room temperature. The mixture was filtered, and the filtratewas diluted with dichloromethane (100 ml) and water (50 ml). The organicsolution was separated, dried using magnesium sulfate, and concentratedin a vacuum. The residue was distilled to obtain 33.3 g of(R)-4-methoxycarbonyl-2-phenyl-1,3-oxazolin-2-en (11). b.p.: 120-123°C./0.09 mm, [α]_(D) ²¹=−118.20° (c=1.13, CHCl₃).

[0150] To a stirred and cooled solution of the(R)-4-methylcarbonyl-2-phenyl-1,3-oxazolin-2-en (11) (1.4 g, 6.8 mmols)in toluene (30 ml) and n-hexane (5 ml), a solution of DIBAL-H (1.7 M,6.0 ml, 10.2 mmols) in n-hexane was added dropwise at −70° C. under anAr gas. The mixture was stirred for 2 hours at −70° C. Then, MeOH (1 ml)was added dropwise at −70° C., and the mixture was stirred for 30minutes. Then, an EtOAc solution (10 ml) and a saturated aqueoussolution (20 ml) of potassium sodium tartrate were added to terminatethe reaction. By removing the cooling bath, the temperature was raisedto room temperature. The mixture was partitioned between EtOAc (500 mL)and a saturated aqueous solution (1.5 L) of potassium sodium tartrate.The organic solution was dried over magnesium sulfate, and thenconcentrated in a vacuum to obtain 1.4 g (quantitative) of(R)-4-formyl-2-phenyl-1,3-oxazolin-2-en (12) as a crude yellow oil.

[0151] (2) Synthesis of the Compound of Example 1

[0152] Step 1: The (R)-4-formyl-2-phenyl-1,3-oxazolin-2-en (12) (1.2 g,about 5.8 mmols) obtained in (1)(B), which was dissolved in ether (5ml), was added to a solution of the alkenylalane (8) obtained in (1)(A),and the mixture was stirred at 0 to 5° C. The temperature was returnedto room temperature, and stirring was continued for 2 hours. The mixturewas poured into a saturated potassium sodium tartrate solution (400 ml),and extracted with EtOAc (400 ml). The EtOAc solution was dried overmagnesium sulfate, and then concentrated in a vacuum. TLC analysis(n-hexane:ether,3:7) of the residue showed it to be a mixture of twocompounds, one of which had Rf of 0.56, the other having Rf of 0.39.These two compounds were subjected to silica gel column chromatography.On elution with n-hexane:ether(3:1), a nonpolar crystalline isomer, 404mg (21.4% from the compound (7)) of (1′R)-isomer (recrystallized fromn-hexane), was obtained first. Further elution with the same solventgave a polar isomer, 294 mg (15.6% from the compound (7)) of acorresponding (1′S)-isomer, namely,(4R,1′S)-4-(1′-hydroxy-7′-methyl-2′,6′-hexadecadienyl)-2-phenyl-1,3-oxazolin-2-enisomer (13). This crystalline isomer being an erythro-isomer wasconfirmed by converting it later into a final product in (4E,8E) form.

[0153] Step 2: 2N—HCl (1 ml) was added to a THF solution (4 ml) of the(4R,1′S)-4-(1′-hydroxy-7′-methyl-2′,6′-hexadecadienyl)-2-phenyl-1,3-oxazolin-2-en(13) (160 mg, 0.4 mmol). The mixture was stirred for 20 hours at roomtemperature. The reaction mixture was diluted with ice water (10 ml),and extracted with CHCl₃-MeOH (87:13, 25 ml×3). The organic solution wasdried over magnesium sulfate, and concentrated in a vacuum to obtainabout 200 mg (quantitative) of a compound (14).

[0154] Step 3: The compound (14) was dissolved in pyridine (1 ml), and apyridine solution (1 ml) of p-nitrophenyl(S)-2-acetoxyhexadecanoate (400mg, 0.92 mmol) was added to the solution, followed by stirring themixture for 20 hours at 45° C. The solvent was removed under vacuum, andthe residue was subjected to silica gel column chromatography. Elutionwith n-hexane:ether(2:1) gave a yellow oil. The small amount of oil inn-hexane solution precipitated crystals of(4E,8E,2R,3S,2′S)-N-2′-acetoxyhexadecanoyl-1-O-benzoyl-9-methyl-4,8-octadecadiene-1,3-diol.Recrystallization of the crystals from n-hexane gave 184 mg of a pureproduct.

[0155] This product (425 mg, 0.6 mmol) was dissolved in CHCl₃ (30 ml),and the solution was added to an MeOH solution (0.3N, 20 ml) of NaOH,followed by stirring the mixture for 15 minutes at room temperature. Themixture was poured into ice-cooled water (100 ml) and extracted withCHCl₃ (300 ml×2). The CHCl₃ solution was washed (a saturated aqueoussolution of sodium chloride), dried (magnesium sulfate), andconcentrated under vacuum, whereafter the residue was subjected tosilica gel column chromatography. Elution with CHCl₃-EtOAC (3:2) gave asolid, which was recrystallized from n-hexane to obtain 248 mg (73.4%)of(4E,8E,2R,3S,2′S)-N-2′-hydroxyhexadecanoyl-9-methyl-4,8-octadecadiene-1,3-diol(15). mp: 62.0-63.0° C., [α]²³ _(D)=−7.3 (c=0.61, CHCl₃)

Example 2 Synthesis of(4E,8E,2R,3S,2′S)-N-2′-hydroxyhexadecanoyl-9-methyl-4.8-octadecadiene-1,3-diol

[0156] (1) Preparation of Reaction Materials

[0157] (A) Synthesis of N-Boc-Protected (R)-Formyloxazolidine Derivative

[0158] An N-Boc-protected (R)-2,2-dimethyl-4-formyloxazolidine wassynthesized from (R)-serine in accordance with a scheme illustratedbelow.

[0159] (B) Synthesis of a Tert-Butyldiphenylsilyl (TBDPS) Protected Acid

[0160] (2S)-2-(tert-butyldiphenylsilyloxy)hexadecanoic acid wassynthesized in accordance with a scheme shown below.

[0161] (2) Synthesis of Starting Materials and the Compound of Example 2

[0162] A synthesis scheme is shown below.

[0163] (A) Synthesis of Starting Materials

[0164] 2-Undecanone (methyl nonyl ketone) (1′) (73.9 g, 434 mmols) andmethyl (diethylphosphono)acetate (99.4 g, 433 mmols) were dissolved indry benzene (300 ml). The solution was stirred at room temperature, anda 28% sodium methoxide solution (83.7 g) in methanol was slowly added.Stirring was continued overnight at room temperature. The reactionmixture was poured into ice water, and extracted with diethyl ether. Theorganic layer was washed (water and a saturated aqueous solution ofsodium chloride), and dried (sodium sulfate), followed by removing thesolvent in a vacuum to obtain methyl 3-methyl-2-dodecenoate (2′) as ageometric isomer mixture.

[0165] A dry THF solution (150 ml) of the crude product, methyl3-methyl-2-dodecenoate (2′) (E/Z mixture, 98.5 g, 0.435 mol), was addeddropwise at room temperature to a stirred suspension of LiAlH₄ (16.5 g,0.435 mol) in dry THF (300 ml). The reaction mixture was heated for 2hours under reflux. After the mixture was reverted to room temperature,water and 10% sulfuric acid were slowly added in sequence, and theresulting mixture was extracted with diethyl ether. The organic layerwas washed with water and a saturated aqueous solution of sodiumchloride, dried over magnesium sulfate, and then concentrated undervacuum to obtain crude (E)-3-methyl-2-dodecen-1-ol (85.5 g, 99%) as anE/Z mixture. The E/Z ratio of the crude alcohol mixture was found to be96:4 as a result of 250 MHz ¹H-NMR analysis. A portion (50 g) of theresidue was subjected to silica gel column chromatography (hexaneelution) to obtain 41.3 g (83%) of a pure E isomer (3′).

[0166] An acetonitrile solution (100 ml) of triphenylphosphine (18.6 g,70.9 mmols) was stirred at 0° C. Bromine (11.4 g, 3.7 ml, 71.3 mmols)was slowly blended into the solution. To the mixture, an acetonitrile(30 ml) solution of (E)-3-methyl-2-dodecen-1-ol (3′) was added dropwise,followed by stirring for 2 hours at 0° C. The solvent was removed undervacuum, and the residue was dissolved in dichloromethane. The solutionwas washed with saturated sodium bicarbonate and a saturated aqueoussolution of sodium chloride, then dried over sodium sulfate, andconcentrated. Pentane was added to the residue, and a solid formed wasremoved by filtration. The filtrate was concentrated in a vacuum toobtain 17.9 g (97%) of (E)-1-bromo-3-methyl-2-dodecene (4′).

[0167] A diethyl ether solution (100 ml) of propargyl bromide (22.4 g,190 mmols) was added dropwise to magnesium (5.20 g, 210 mmols) and HgCl₂(360 mg, 1.33 mmols) to obtain propargyl magnesium bromide (Grignardreagent). CuCl (200 mg, 2.02 mmols) was added to this Grignard reagent,and the resulting solution was ice-cooled. After a diethyl ether (100ml) solution of (E)-1-bromo-3-methyl-2-dodecene (4′) (18.8 g, 72.0mmols) was added dropwise, the mixture was stirred for 3 hours at 0° C.The reaction mixture was poured into ice water, acidified with dilutedhydrochloric acid, and extracted several times with diethyl ether. Theorganic layers were combined, and washed with water and a saturatedaqueous solution of sodium chloride. A small amount of allene typeimpurities was removed by silica gel column chromatography (elution withhexane) to obtain pure (E)-6-methyl-5-pentadecen-1-yne (5′) (12.7 g,80%).

[0168] (B) Synthesis of the Compound of Example 2

[0169] Step 1: An n-hexane solution of n-butyl lithium (1.68 M, 10 ml,16.8 mmols) was added at −23° C. to a dry THF (50 ml) solution of the(E)-6-methyl-5-pentadecen-1-yne (5′) (4.0 g, 18.2 mmols) synthesized in(1)(B). Then, the mixture was stirred for 1 hour at the same temperatureunder an argon gas. A dry THF (30 ml) solution of the N-Boc-protected(R)-2,2-dimethyl-4-formyloxazolidine (3.7 g, 16.1 mmols) synthesized in(1)(A) was blended at −23° C. into the stirred mixture, and then theresulting mixture was stirred for 3 hours at the same temperature.Subsequently, the reaction mixture was poured into ice water, andextracted several times with diethyl ether. The organic extractscombined were washed (water and a saturated aqueous solution of sodiumchloride), dried (magnesium sulfate), and concentrated in a vacuum. Theresulting light yellow oily matter was purified by silica gel columnchromatography (eluted with hexane:AcOEt=20:1) to obtain 5.17 g (11.5mmols, 71%) of tert-butyl(4R,1′S)-4-(1′-hydroxy-7′-methyl-6′-hexadecen-2′-ynyl)-2,2-dimethyl-3-oxazolidinecarboxylate(6′).

[0170] Step 2: A dry THF (100 ml) solution of the tert-butyl(4R,1′S)-4-(1′-hydroxy-7′-methyl-6′-hexadecen-2′-ynyl)-2,2-dimethyl-3-oxazolidinecarboxylate(6′) (8.4 g, 18.7 mmols) was added dropwise to an ethylamine (50 g) bluesolution of lithium (2 g, 288 mmols) over 1 hour with stirring at −70°C. After stirring was continued for 4 hours at −70° C., the mixture wasreturned gradually to the ambient temperature, and treated with asaturated ammonium chloride solution. Ethylamine and the solvent wereremoved in a vacuum, and water was added to the residue. The mixture wasextracted several times with diethyl ether. The organic extractscombined were washed with a saturated aqueous solution of sodiumchloride, dried over sodium sulfate, and concentrated in a vacuum toobtain 4.5 g (14.4 mmols, 77%) of crude(4E,8E,2R,3S)-9-methyl-2-amino-4,8-octadecadiene-1,3-diol (7′) as abrown oil.

[0171] Step 3: A mixture of the crude(4E,8E,2R,3S)-9-methyl-2-amino-4,8-octadecadiene-1,3-diol (7′) (4.3 g,13.8 mmols), pyridinium p-toluenesulfonate (PPTS) (3.47 g, 13.8 mmols),and 2,2-dimethoxypropane (20 ml) in trichloromethane (120 ml) was heatedfor 4 hours under reflux. The mixture was cooled to room temperature,and diluted with trichloromethane. The dilution was washed (a saturatedsolution of sodium hydrogen carbonate, water, and a saturated aqueoussolution of sodium chloride), then dried (sodium sulfate), andconcentrated in a vacuum. The residue was purified by silica gelchromatography (eluted with CH₂Cl₂:MeOH=50:1) to obtain 4.10 g of(4E,8E,2R,3S)-2-amino-1,3-O-isopropylidene-9-methyl-4,8-octadecadiene(8′) as a light brown oil (yield 88% based on the 6′ compound). n_(D)²²: 1.4751, [α]²⁴ _(D)=−8.78 (c=1.85, CHCl₃).

[0172] The (2S)-2-(tert-butyldiphenylsilyloxy)hexadecanoic acid (2.20 g,4.10 mmols) synthesized in (1)(B), dicyclohexylcarbodiimide (DCC, 850mg, 4.1 mmols), and 1-hydroxybenzotriazole (HOBt) (555 mg, 4.10 mmols)were dissolved in dry dichloromethane (40 ml). With the solution beingstirred at room temperature, a dry dichloromethane solution (20 ml) ofthe(4E,8E,2R,3S)-2-amino-1,3-O-isopropylidene-9-methyl-4,8-octadecadiene(8′) (1.4 g, 4.1 mmols) was added dropwise. The reaction mixture wasstirred for 2 hours at room temperature, and then concentrated in avacuum to a half amount, and the resulting urea was removed byfiltration through Celite.

[0173] The filtrate was washed (a saturated solution of sodium15-hydrogen carbonate, water, and a saturated aqueous solution of sodiumchloride), dried (magnesium sulfate), and concentrated in a vacuum. Theresidue was purified by silica gel column chromatography (eluted withhexane:AcOEt,50:1) to obtain 1.82 g (51%) of(4E,8E,2R,3S,2′S)-2-[2′-(OTBDPS)hexadecanoylamino]-1,3-O-(isopropylidenedioxy)-9-methyl-4,8-octadecadiene(9′).

[0174] Step 4: The(4E,8E,2R,3S,2′S)-2-[2′-(OTBDPS)hexadecanoylamino]-1,3-O-(isopropylidenedioxy)-9-methyl-4,8-octadecadiene(9′) (1.1 g, 1.26 mmols) was dissolved in CH₂Cl₂:MeOH (1:1, 20 ml).Pyridinium p-toluenesulfonate (PPTS, 320 mg) was added to the solution,the mixture was stirred for 1 hour at room temperature, and the solventwas removed in a vacuum. The residue was dissolved in AcOEt, and thenthe solution was washed (a saturated solution of sodium hydrogencarbonate, water, and a saturated aqueous solution of sodium chloride),dried (magnesium sulfate), and concentrated in a vacuum. The residue waspurified by silica gel column chromatography to obtain(4E,8E,2R,3S,2′S)-2-[2′-(OTBDPS)hexadecanoylamino]-9-methyl-4,8-octadecadiene-1,3-diol(10′) (680 mg, 65%).

[0175] The TBDPS ether,(4E,8E,2R,3S,2′S)-[2-(2′-(OTBDPS)hexadecanoylamino]-9-methyl-4,8-octadecadiene-1,3-diol(10′) (640 mg, 0.71 mmol), was dissolved in THF (50 ml).Tetra-n-butylammonium fluoride (1M THF solution, 1.2 ml, 1.2 mmols) wasadded to the solution, and the mixture was stirred for 1 hour at roomtemperature. The reaction mixture was poured into water, and extractedwith dichloromethane. The organic layer was washed with water and asaturated aqueous solution of sodium chloride, then dried over magnesiumsulfate, and concentrated in a vacuum. The residue was purified bysilica gel column chromatography (eluted with hexane:AcOEt, 1:1), andrecrystallized from acetone to obtain(4E,8E,2R,3S,2′S)-N-2′-hydroxyhexadecanoyl-9-methyl-4,8-octadecadiene-1,3-diol(11′) (424 mg, 93%). mp: 82.0° C., [α]²⁴ _(D): +8.2 (c=1.0, CHCl₃)

Example 3 Synthesis of(4E,2S,3R,2′RS)-N-2′-hydroxyhexadecanoyl-9-methyl-4-octadecene-1,3-diol

[0176] (1) Preparation of Reaction Materials

[0177] (A) Synthesis of N-Boc-Protected (S)-Formyloxazolidine Derivative

[0178] An N-Boc-protected (S)-2,2-dimethyl-4-formyloxazolidine wassynthesized from (S)-serine in accordance with a scheme illustratedbelow.

[0179] (B) Synthesis of a Tert-Butyldiphenylsilyl (TBDPS) Protected Acid

[0180] (2RS)-2-(tert-butyldiphenylsilyloxy)hexadecanoic acid wassynthesized in accordance with a scheme shown below.

[0181] (2) Synthesis of Example 3

[0182] A synthesis scheme is shown below.

[0183] Step 1: Pd-C (1.0 g) was added to an ethyl acetate solution (300ml) of 3-methyl-2-dodecen-1-ol (3″) (30 g, 0.15 mol) obtained from2-undecanone (1″) in the same manner as in Example 2, and the mixturewas stirred for 3 days in a hydrogen atmosphere. The reaction mixturewas filtered through Celite, and the filtrate obtained was concentratedand then distilled under reduced pressure to obtain 3-methyldodecan-1-ol(4″) (20 g, 67%). b.p. 122-123° C./4 torrs. ¹H-NMR (90 MHz, CDCl₃)0.8-1.0 (6H,m,Me), 1.0-1.7 (20H,m,2˜11-H and OH), 3.66 (2H,q,J=7,1-H).

[0184] Step 2: Pyridine (20 ml) was added to a methylene chloride (50ml) solution of the 3-methyldodecan-1-ol (4″) (12.8 g, 63.9 mols), andp-toluenesulfonyl chloride (12.8 g, 67.1 mmols) was further added underice-cooling. The reaction mixture was stirred overnight at 4° C., thenpoured into diluted hydrochloric acid, and extracted with hexane. Theorganic layer was washed (water, a saturated aqueous solution of sodiumbicarbonate, a saturated aqueous solution of sodium chloride), dried(magnesium sulfate), and concentrated under reduced pressure to obtain21.7 g of a tosylate. Sodium bromide (9.9 g, 96 mols) was added to a DMFsolution (100 ml) of the resulting tosylate, and the mixture was stirredovernight at room temperature. The reaction mixture was poured intowater, and extracted with hexane. The organic layer was washed (waterand a saturated aqueous solution of sodium chloride), dried (magnesiumsulfate), and concentrated under reduced pressure to obtain1-bromo-3-methyldodecane (5″) (15.1 g, 90%). ¹H-NMR (90 MHz, CDCl₃)0.8-1.0 (6H,m,Me), 1.0-2.0 (19H,m,2˜11-H), 3.43 (2H,br t,J=7,1-H).

[0185] Step 3: Tetramethylethylenediamine (TMEDA) (15 ml) was added to aTHF (80 ml) solution of propyne (about 4 g, 0.1 mol) under argon. To themixture, n-BuLi (1.55 M, 64.5 ml, 100 mmols) was added dropwise at −78°C. With the temperature being raised gradually to 0° C., the mixture wasstirred for 1.5 hours, and then cooled again to −78° C. Thereto, anHMPA-THF (20 ml+20 ml) solution of the 1-bromo-3-methyldodecane (5″)(13.2 g, 50 mmols) was added dropwise, and with the temperature of themixture being raised gradually to room temperature, stirring wascontinued overnight. The reaction mixture was poured into a saturatedaqueous solution of ammonium chloride, and extracted with hexane. Theresulting organic layer was washed (water, a saturated aqueous solutionof sodium bicarbonate, a saturated aqueous solution of sodium chloride),dried (magnesium sulfate), and concentrated under reduced pressure. Theresulting residue was purified by silica gel column chromatography toobtain 6-methyl-2-pentadecyne (6″) (12.1 g, 97%). ¹H-NMR (90 MHz, CDCl₃)0.84 (3H,d,J=7,6-Me), 0.88 (3H,t,J=7,15-H), 1.0-1.6 (19H,m,5˜14-H), 1.77(3H,t,J=2.5,1-H), 2.12 (2H,m,4-H).

[0186] Step 4: To anhydrous 1,3-diaminopropane (180 ml), which had beendistilled, metallic lithium (2.8 g, 0.4 mol) was added under argonstream, and the mixture was stirred for 2 hours at 70° C. After themixture was allowed to cool, potassium t-butoxide (27 g, 0.24 mol) wasadded, followed by stirring for 15 minutes. The 6-methyl-2-pentadecyne(6″) (12.1 g, 54.5 mmols) was added dropwise, and the mixture wasstirred overnight at room temperature. This reaction was carefullyquenched with a saturated aqueous solution of ammonium chloride, andthen the mixture was extracted with ether. The resulting organic layerwas washed (diluted hydrochloric acid, a saturated aqueous solution ofsodium bicarbonate, a saturated aqueous solution of sodium chloride),dried (magnesium sulfate), and concentrated under reduced pressure. Theresulting residue was purified by silica gel column chromatography toobtain 6-methyl-1-pentadecyne (7″) (9.90 g, 82%). IR and ¹H-NMR analysisof the product showed the following results: IR (film) 3300 (m,C≡CH),2130 cm⁻¹ (w,C≡C). ¹H-NMR (90 MHz, CDCl₃) 0.84 (3H,d,J=7,6-Me), 0.88(3H,t,J=7,15-H), 1.0-1.6 (21H,m,4˜14-H), 1.93 (1H,t,J=2.5,1-H), 2.0-2.3(2H,m,3-H).

[0187] Step 5: An n-hexane solution (1.68 M, 10 ml, 16.8 mmols) ofn-butyl lithium was added at −23° C. to a dry THF solution of the6-methyl-1-pentadecyne (7″) (4.0 g, 18.2 mmols). Then, the mixture wasstirred for 1 hour at the same temperature under an argon gas. A dry THF(30 ml) solution of the N-Boc-protected(S)-2,2-dimethyl-4-formyloxazolidine (3.7 g, 16.1 mmols) synthesized inthe above (1)(A) was blended at −23° C. into the stirred mixture, andthen the resulting mixture was stirred for 3 hours at the sametemperature. Subsequently, the reaction mixture was poured into icewater, and extracted several times with diethyl ether. The organicextracts combined were washed (water and a saturated aqueous solution ofsodium chloride), dried (magnesium sulfate), and concentrated in avacuum. The resulting light yellow oily matter was purified by silicagel column chromatography (eluted with hexane:AcOEt=20:1) to obtain 5.17g (11.5 mmols, 71%) of tert-butyl(4S,1′R)-4-(1′-hydroxy-7′-methylhexadecan-2′-ynyl)-2,2-dimethyl-3-oxazolidinecarboxylate(8″).

[0188] Step 6: A dry THF (100 ml) solution of the tert-butyl(4S,1′R)-4-(1′-hydroxy-7′-methylhexadecan-2′-ynyl)-2,2-dimethyl-3-oxazolidinecarboxylate(8″) (8.4 g, 18.7 mmols) was added dropwise to an ethylamine (50 g) bluesolution of lithium (2 g, 288 mmols) over 1 hour with stirring at −70°C. After stirring was continued for 4 hours at −70° C., the mixture wasreturned gradually to the ambient temperature, and treated with asaturated ammonium chloride solution. Ethylamine and the solvent wereremoved in a vacuum, and water was added to the residue. The mixture wasextracted several times with diethyl ether. The organic extractscombined were washed with a saturated aqueous solution of sodiumchloride, dried over sodium sulfate, and concentrated in a vacuum toobtain 4.5 g (14.4 mmols, 77%) of crude(4E,2S,3R)-9-methyl-2-amino-4-octadecene-1,3-diol (9″) as a brown oil.

[0189] Step 7: A mixture of the crude(4E,2S,3R)-9-methyl-2-amino-4-octadecene-1,3-diol (9″) (4.3 g, 13.8mmols), pyridinium p-toluenesulfonate (PPTS) (3.47 g, 13.8 mmols), and2,2-dimethoxypropane (20 ml) in trichloromethane (120 ml) was heated for4 hours under reflux. The mixture was cooled to room temperature, anddiluted with trichloromethane. The dilution was washed (a saturatedsolution of sodium hydrogen carbonate, water, and a saturated aqueoussolution of sodium chloride), then dried (sodium sulfate), andconcentrated in a vacuum. The residue was purified by silica gelchromatography (CH₂Cl₂:MeOH=50:1) to obtain 4.10 g of(4E,2S,3R)-2-amino-1,3-O-isopropylidene-9-methyl-4-octadecene (10″) as alight brown oil (yield 88% based on the 8″ compound).

[0190] The (2RS)-2-(tert-butyldiphenylsilyloxy)hexadecanoic acidsynthesized in (1)(B) (2.20 g, 4.10 mmols), dicyclohexylcarbodiimide(DCC, 850 mg, 4.1 mmols), and 1-hydroxybenzotriazole (HOBt) (555 mg,4.10 mmols) were dissolved in dry dichloromethane (40 ml). With thesolution being stirred at room temperature, a dry dichloromethanesolution (20 ml) of the(4E,2S,3R)-2-amino-1,3-O-isopropylidene-9-methyl-4-octadecene (10″) (1.4g, 4.1 mmols) was added dropwise. The reaction mixture was stirred for 2hours at room temperature, and then concentrated in a vacuum to a halfamount, and the resulting urea was removed by filtration through Celite.The filtrate was washed (a saturated solution of sodium hydrogencarbonate, water, and a saturated aqueous solution of sodium chloride),dried (magnesium sulfate), and concentrated in a vacuum. The residue waspurified by silica gel column chromatography (eluted withhexane:AcOEt,50:1) to obtain 1.82 g (51%) of(4E,2S,3R,2′RS)-2-[2′-(OTBDPS)hexadecanoylamino]-1,3-O-(isopropylidenedioxy)-9-methyl-4-octadecene(11″).

[0191] Step 8: The(4E,2S,3R,2′RS)-2-[2′-(OTBDPS)hexadecanoylamino]-1,3-O-(isopropylidenedioxy)-9-methyl-4-octadecene(11″) (1.1 g, 1.26 mmols) was dissolved in CH₂Cl₂:MeOH (1:1, 20 ml).Pyridinium p-toluenesulfonate (PPTS, 320 mg) was added to the solution,the mixture was stirred for 1 hour at room temperature, and the solventwas removed in a vacuum. The residue was dissolved in AcOEt, then thesolution was washed (saturated sodium hydrogen carbonate, water, and asaturated aqueous solution of sodium chloride), dried (magnesiumsulfate), and concentrated in a vacuum. The residue was purified bysilica gel column chromatography to obtain(4E,2S,3R,2′RS)-2-[2′-(OTBDPS)hexadecanoylamino]-9-methyl-4-octadecene-1,3-diol(12″) (680 mg, 65%).

[0192] The TBDPS ether,(4E,2S,3R,2′RS)-2-[2′-(OTBDPS)hexadecanoylamino]-9-methyl-4-octadecene-1,3-diol(640 mg, 0.71 mmol), was dissolved in THF (50 ml). Tetra-n-butylammoniumfluoride (1M THF solution, 1.2 ml, 1.2 mmols) was added to the solution,and the mixture was stirred for 1 hour at room temperature. The reactionmixture was poured into water, and extracted with dichloromethane. Theorganic layer was washed (water and a saturated aqueous solution ofsodium chloride), then dried (magnesium sulfate), and concentrated in avacuum. The residue was purified by silica gel column chromatography(hexane:AcOEt, 1:1), and recrystallized from acetone to obtain(4E,2S,3R,2′RS)-N-2′-hydroxyhexadecanoyl-9-methyl-4-octadecene-1,3-diol(13″) (400 mg, 89%). mp: 52-57° C., [α]²⁰ _(D): +3.7 (c=0.06, CHCl₃)

Example 4 Edg Receptor Response Test

[0193] HL 60 cells were obtained from a cell bank, and subcultured forabout 50 passages over a half-year period in accordance with the methoddescribed in BBRC '98, 263, p. 253 using an RPMI-1640 culture medium(Gibco) containing 10% fetal bovine serum to prepare a premyeloblastomacell strain HL60 expressing Edg receptors on the cell surface. Using thepremyeloblastoma cell strain HL60 expressing Edg receptors on the cellsurface, the cell response of test substances was investigated. Anincrease in the intracellular Ca²⁺ concentration was measured as anindicator of cell response. It is reported that when the Edg receptor onthe HL60 cell surface is bound to AHOP, it phosphorylates G protein toactivate IP₃ kinase, whereafter the intracellular Ca²⁺ concentrationincreases (FEBS Letter '96, 379, p. 260, BBRC '98, 253, p. 253). Thus,the intracellular Ca²⁺ concentration serves as an indicator of Edgreceptor response.

[0194] A Ca²⁺ chelating reagent, Fura-2AM, was taken into HL60 cells.

[0195] A cell suspension (1.2 ml) was charged into a quartz cell, whichwas then mounted on a fluorometer LS50B (Perkin Elmer, for cellmeasurement). An excitation wavelength was alternately switched between340 nm (exciting Fura-2 having chelated Ca²⁺) and 380 nm (excitingunreacted Fura-2) at intervals of 0.5 milli second, and fluorescenceintensity at 510 nm was measured.

[0196] Each test substance was added at an end concentration of 30 μM bymeans of a microsyringe, and then fluorescence intensity was traced toinvestigate whether Ca²⁺ would increase or not. It was also confirmedwhether Ca²⁺ would increase or not when AHOP (1 μM) was added afteraddition of the test substance. Through these tests, the AHOP antagonismof each substance was studied.

[0197] After the compound of Example 2 or the compound of Example 3 wasadded, the increase in the intracellular Ca²⁺ concentration by theaddition of AHOP was inhibited. This finding suggested the possibilitythat these two substances were Edg antagonistic.

[0198] Then, a study was made of the dose dependency of theintracellular Ca²⁺ increase inhibiting action of the two substancessuggested to have antagonism, the compound of Example 2 and the compoundof Example 3. Suramin, already confirmed to have antagonistic action,was used as a comparison. The experimental procedure was the same asdescribed above, except that AHOP (1 μM) was added after addition of thetest substance at varying concentration. An increase in the Ca²⁺concentration was evaluated by an increased Ca²⁺ concentration (%) as avalue relative to the increased Ca²⁺ concentration in a negative controlgroup (no drug added).

[0199] As a result, the compound of Example 3 at a concentration of 0.3to 3 μM, and the compound of Example 2 at a concentration of 0.03 to 0.3μM, dose-dependently suppressed the Ca²⁺ concentration increase by AHOP.The results are shown in FIG. 1.

[0200] A 50% inhibitory concentration (ED₅₀) for the intracellular Ca²⁺concentration increase was 1.2±0.1 μM for the compound of Example 3, and0.041±0.1 μM for the compound of Example 2. The ED₅₀ of suramin, whoseEdg antagonism was already reported, is 1.8±0.1 μM. In comparison, thestrength of the action that the compound of Example 2 has is about 40times as high. These ED₅₀ values are shown in Table 1. TABLE 1 50%Inhibition Point for Ca²⁺ Increase Substance ED₅₀ (μM) Positive controlsuramin 1.8 ± 0.1 Test substance Example 2 0.041 ± 0.1  Example 3 1.2 ±0.1

Example 5 Competition Experiments Using ³H-AHOP

[0201] The same premyeloblastoma cell strain HL60 expressing Edgreceptors on the cell surface as used in Example 4 was used. The cellswere harvested by centrifugation, then suspended in an F-12 culturemedium (stored at 4° C., 10 ml), and carried into an RI laboratory.³H-AHOP (15 μCi/1 nM) at an end concentration of 1 nM, and an unlabeledcompound (the compound of Example 2 or Example 3) at an endconcentration of 10 nM, 30 nM or 100 nM were each added to 200 μl of thecell suspension (1×10⁶ cells/ml F-12), and a binding test was conductedfor 30 minutes at 4° C. (with occasional stirring). After centrifugationfor 7 minutes at 12,000 rpm, the supernatant was rapidly (withoutdamaging cell pellets) discarded with a micropipetter. The cell pelletswere suspended in 1.5 ml of Ready Safe (Beckman), whereafter thesuspension was transferred into a vial and measured for radioactivity bya liquid scintillation counter L2100 (Beckman).

[0202] As a result, the compounds of Example 2 and Example 3 werecompetitive with ³H-AHOP, as with AHOP. Thus, these two substances wereconsidered to bind to Edg specifically.

[0203] The compounds of Example 2 and Example 3 were subjected to thesame competition experiments as described above, except that they wereadded at concentrations of 1 nM, 3 nM and 10 nM. Dose-dependentinhibition of binding of ³H-AHOP (to HL60 cells) was observed. Theresults are shown in FIG. 2.

Example 6 Action on Vascular Smooth Muscle

[0204] The action of the test substances on vascular smooth musclegrowth was investigated.

[0205] It is hypothesized that with the progression of arteriosclerosis,vascular smooth muscle cells are transformed from the contractile typeto the synthetic type, and while secreting inflammatory cytokines, thevascular smooth muscle cells proliferate, causing arterioscleroticlesions to proceed (Roth's hypothesis). There is a report that Edgreceptors are expressed on the surface of vascular smooth muscle cells(The American Society for Pharmacology and Experimental Therapeutics'00, Vol. 58, p. 449; Vascular smooth muscle cells are reported toproliferate in response to sphingosylphosphorylcholine which acts on Edgreceptors like AHOP (The American Physiological Society '98, C1255)).

[0206] Thus, the actions of the compounds of Examples 2 and 3 on thegrowth of vascular smooth muscle cells were measured in the mannerdescribed below. Suramin confirmed to have Edg receptor antagonism wasused as a positive control.

[0207] The rat carotid intima was rubbed by ballooning, and a tissuefragment was cultured by explant culture. Two weeks later, vascularsmooth muscle cells harvested were cultured in a DMEM culture medium(Gibco) containing 10% fetal bovine serum. The cultures were subculturedseveral times for stabilization, whereafter the subcultures were seededat a cell density of 5×10³ cells/cm² for use in experiments.

[0208] Along with the growth factor sphingosylphosphorylcholine (10 μM),the compound of Example 2 or 3, or suramin was added to the cells, and24 hours later, the cell density was measured by BrdU assay (Science'82, 218, p. 474, Cytometry '85, 6, p. 584).

[0209] As a result, the compounds of Examples 2 and 3 inhibited vascularsmooth muscle growth dose-dependently at concentrations of 0.3 to 3 μMand 1 to 10 μM, respectively. Suramin, used as the positive control,inhibited vascular smooth muscle growth at concentrations of 30 and 100μM. The results are shown in FIG. 3.

Example 7 Anti-Inflammatory Tests Using Pseudo-Blood Vessel Model

[0210] At the site of injury in vivo, the exposed collagen(extracellular matrix) is targeted as an injury signal, and plateletsare aggregated there. Inflammatory cytokines (such as PDGF) releasedfrom the aggregated and activated platelets advance inflammation.Moreover, severe inflammation is presumed to destroy homeostasis ofcardiovascular organs and progress arteriosclerosis. AHOP is alsoconsidered to have the same action as PDGF.

[0211] Hence, AHOP was used as an inflammation-inducing agent toestablish a pseudo-blood vessel in vitro model. Using the model, it wasstudied whether the compounds of the present invention showanti-inflammatory action, and thereby have possibilities for maintainingthe homeostasis of cardiovascular organs and acting in a directiontoward improvement of pathophysiological states.

[0212] (1) Inflammation-Inducing Action of AHOP in Pseudo-Blood VesselModel

[0213] Transwells were used, each consisting of an upper compartmentseparated from a lower compartment by a porous membrane. A single layerof bovine endothelial cells was cultured on the porous membrane at thebottom surface of the transwell upper compartment. A suspension offluorescence-labeled neutrophils was added to the transwell uppercompartment, and AHOP was suspended within the lower compartment to anend concentration of 0.1 to 10 microM. That is, a pseudo-blood vessel invitro inflammation model was thus constructed in which the uppercompartment and the lower compartment of the transwell were isolatedfrom each other via the endothelial layer, and the upper compartmentcorresponded to the interior of a blood vessel, while the lowercompartment corresponded to the site of inflammation outside the bloodvessel. Measurements were made of the number of the neutrophils passingfrom the upper compartment into the lower compartment through theendothelial layer, and the number of the neutrophils adhering to theendothelial layer. At an AHOP concentration of 10 microM, thetransmigration through the endothelial layer and the adhesion of theneutrophils were promoted significantly. That is, AHOP was considered toact as an inflammation-inducing substance.

[0214] (2) Action of the Compounds (Edg antagonists) of the PresentInvention on Inflammatory Cell-Vascular Endothelial Cell Interaction

[0215] AHOP was used as an inflammation inducer, and the effect of thecompounds of Examples 2 and 3, showing Edg antagonism, on thepseudo-blood vessel in vitro inflammation model was investigated.

[0216] That is, the compound of Example 2 or 3 was added in an amount of0.01 to 1 microM to the upper compartment or lower compartment of thetranswell, and 10 microM AHOP was placed in the lower compartment toinduce inflammation. As a control, inflammation was induced in the sameway as above but without addition of any compounds of the invention.

[0217] Measurements were made of the number of the neutrophils passingfrom the upper compartment into the lower compartment through theendothelial layer, and the number of the neutrophils adhering to theendothelial layer. A relative neutrophil count (%) was calculated fromthe following equation:

Relative neutrophil count (%)=[number of neutrophils (passing andadhering) in the experimental group]/[number of neutrophils (passing andadhering) in the control]×100

[0218] The results are shown in FIG. 4. As shown in the drawing,neutrophil transmigration and adhesion were suppressed by 0.1 and 1microM of the compounds of Examples 2 and 3.

[0219] Hence, when AHOP is used as an inflammation-inducing agent in thepseudo-blood vessel in vitro model, the compounds of Examples 2 and 3are assumed to exert anti-inflammatory action, and thereby havepossibilities for maintaining the homeostasis of cardiovascular organsand acting in a direction toward improvement of pathophysiologicalstates.

Example 8 Ligation-Associated Myocardial Infarction Model

[0220] The compound of Example 2 was used as a test substance forinvestigating its effect on myocardial infarction due to reperfusionfollowing ligation of the rabbit coronary artery.

[0221] Male NZW rabbits (weighing 2.83 to 3.20 kg) were purchased fromKitayama Labes, Co. Ltd., and bred and raised under the conditions: roomtemperature 20-26° C., humidity 40-70%, and illumination time 12hours/day (7-19:00). The animals were allowed food and water ad libitum,and quarantined and acclimatized for 2 weeks or more. Then, the animalsin good health were used.

[0222] The above rabbits were administered 10 mg/kg of the compound ofExample 2 through the jugular vein under anesthesia, and then thecompound of Example 2 was continuously infused in a dose of 6.9 microg/kg/min. In a control group, physiological saline (hereinafter referredto as PS) was administered in the same manner as in the experimentalgroup. Then, coronary artery was ligated for 30 minutes, whereafter theligature was released for reperfusion, and the blood pressure in thecarotid artery, the pulse rate and the number of arrhythmias weremeasured. The carotid arterial blood pressure and pulse rate weremeasured before administration, during continuous intravenous infusion,during the ligation period (15 minute later and 30 minutes later), andduring reperfusion. The carotid arterial blood pressure was calculatedas mean blood pressure. The number of arrhythmias was counted as thenumber of extrasystoles that appeared during the ligation period (for 30minutes) or during reperfusion. The results are shown in Tables 2 and 3.

[0223] After 3 hours of reperfusion, the heart was removed, and slicedinto 6 pieces. The living tissues were stained with2,3,5-triphenyltetrazolium hydrochloride (TTC), the area of the infarctdue to ligation was measured, and the percent of the infarct withrespect to the area of the left ventricle was calculated. The resultsare shown in Table 4. TABLE 2 Effects of the compound of Example 2 onblood pressure and pulse rate No. Before Continued Ligation Reperfusionof adminis- i.v. (min) (min) Item Drug animals tration infusion 15 30180 Mean blood PS 4  74 ± 3  75 ± 4  54 ± 8  64 ± 4  68 ± 3 pressure Ex.2 4  83 ± 4  74 ± 9  59 ± 10  66 ± 8  68 ± 4 (mmHg) Pulse rate PS 4 299± 12 293 ± 10 255 ± 24 269 ± 18 269 ± 14 (beats/min) Ex. 2 4 301 ± 9 283± 10 270 ± 9 282 ± 4 256 ± 10

[0224] TABLE 3 Effect of the compound of Example 2 on the number ofarrhythmias Drug No. of animals Ligation Reperfusion PS 4 22 ± 10 19 ±12 Ex. 2 4 12 ± 5  17 ± 6 

[0225] TABLE 4 Effect of the compound of Example 2 on the percent areaof the infarct Infarct/left ventricle Drug No. of animals (%) PS 4 17.9± 2.4 Ex. 2 4 14.0 ± 2.1

[0226] The compound of Example 2 tended to inhibit theligation-associated decrease in pulse rate and reduce the number ofarrhythmias due to ligation in rabbit acute myocardial infarctionmodels. The compound of Example 2 also showed a tendency towarddecreasing the percent area of the infarct.

INDUSTRIAL APPLICABILITY

[0227] The compounds of the present invention show an excellent Edgreceptor antagonizing action. Pharmaceuticals comprising the compoundsof the present invention as an active ingredient exert excellenttherapeutic effects on cardiovascular diseases (e.g. arteriosclerosis,cardiac diseases), cancer, rheumatism, diabetic retinopathy, andrespiratory diseases.

1. An aliphatic compound represented by the following formula (I) orpharmacologically acceptable salts thereof:

where n denotes an integer of 1 to 11, and l denotes an integer of 1 to16, said aliphatic compound being an optical isomer of a (2R,3S,2′S)configuration when an 8-position thereof is a double bond, or an opticalisomer of a (2S,3R,2′RS) configuration when the 8-position is a singlebond.
 2. The compound represented by the formula (I) orpharmacologically acceptable salts thereof according to claim 1, saidcompound being a compound of the following formula (II):

where l and n are as defined in claim
 1. 3. The compound represented bythe formula (I) or pharmacologically acceptable salts thereof accordingto claim 1, said compound being a compound of the following formula(III):

where l and n are as defined in claim
 1. 4. The compound represented bythe formula (I) or pharmacologically acceptable salts thereof accordingto claim 1, said compound being a compound of the following formula(IV):


5. The compound represented by the formula (I) or pharmacologicallyacceptable salts thereof according to claim 1, said compound being acompound of the following formula (V):


6. A method for producing the compound of the formula (I) according toclaim 1, comprising the steps of: (1) reacting an alkenylalane in(E)-form of the following formula: H₃C—(CH₂)_(n)—C(CH₃)

C(H)—(CH₂)₂—CH═CH—Al(R)₂ where n is as defined in claim 1, and R denotesan alkyl group, with an oxazoline aldehyde derivative of the followingformula, having the same optical isomerism as the 2-position of thedesired compound:

where R′ represents an alkyl group or an aryl group; (2) ring-openingthe oxazoline produced in the step (1) to obtain a compound having anNH₂ group and an —OC(═O)R′ group; and (3) N-acylating the product of thestep (2) with an acylating agent having the same optical isomerism asthe 2′-position of the desired compound, and then eliminating a —C(═O)R′group.
 7. A method for producing the compound according to claim 2,comprising the steps of: (1) reacting ofHC≡C—(CH₂)₂—CH═C(CH₃)—(CH₂)_(n)CH₃ in (E)-form (where n is as defined inclaim 2) with an N-protected (R)-formyloxazolidine derivative of thefollowing formula:

where A is a protective group for N, and B and C each represent an alkylgroup; (2) converting the triple bond of the product of the step (1)into an (E)-form double bond, and simultaneously deprotecting theoxazolidine at it undergoes ring-opening, thereby obtaining a compoundhaving an NH₂ group and an OH group; (3) protecting the hydroxyl groupof the product of the step (2), and reacting the protected compound witha compound of the following formula:

where R″ is a protective group for OH, and 1 is as defined in claim 2;and (4) deprotecting the hydroxyl group and eliminating the R″ group. 8.A method for producing the compound according to claim 3, comprising thesteps of: (1) saturating an unsaturated moiety ofHO—CH₂—CH═C(CH₃)—(CH₂)_(n)CH₃ (where n is as defined in claim 3) bycatalytic reduction; (2) substituting a hydroxyl group of the product ofthe step (1) by bromine; (3) substituting the bromine of the product ofthe step (2) by CH₃—C≡C—; (4) shifting the position of a triple bond ofthe product of the step (3) to a terminal thereof to obtain a compoundterminated with the triple bond; (5) reacting said compound terminatedwith the triple bond with an N-protected (S)-formyloxazolidinederivative of the following formula:

where A is a protective group for N, and B and C each represent an alkylgroup; (6) converting the triple bond of the product of the step (5)into an (E)-form double bond, and simultaneously deprotecting theoxazolidine as it undergoes ring-opening, thereby obtaining a compoundhaving an NH₂ group and an OH group; (7) protecting the hydroxyl groupof the product of the step (6), and reacting the protected compound witha compound of the following formula:

where R″ is a protective group for OH, and l is as defined in claim 3;and (8) removing the protective group for the hydroxyl group andeliminating the R″ group.
 9. A pharmaceutical antagonizing endothelialdifferentiation gene (Edg) receptor, said pharmaceutical comprising thecompound or pharmacologically acceptable salts thereof according to anyone of claims 1 to 5 as an active ingredient.
 10. The pharmaceuticalaccording to claim 9 for treatment of cardiovascular disease.
 11. Thepharmaceutical according to claim 10, wherein the cardiovascular diseaseis arteriosclerosis.
 12. The pharmaceutical according to claim 9 fortreatment of cancer.
 13. The pharmaceutical according to claim 9 fortreatment of rheumatism.
 14. The pharmaceutical according to claim 9 fortreatment of diabetic retinopathy.
 15. The pharmaceutical according toclaim 9 for treatment of respiratory disease.
 16. The pharmaceuticalaccording to claim 10, wherein the cardiovascular disease is cardiacdisease.